Osteocalcin (OC) is the most abundant non-collagenous protein in bone, but its function and regulation remain poorly understood. The protein appears to be produced exclusively in bone osteoblasts and chondrocytes and is regulated by vitamin D, retinoic acid and other bone active agents. Clarification of the molecular basis for hormonal and bone-specific expression would greatly improve our understanding of OC's role in normal osteoblast biology. In this project, the applicants will continue a productive line of investigation aimed at identifying the bone-specific elements (BSEs) in the human OC gene. Aim 1 proposes to compare the developmental appearance of the endogenous mouse OC in bone tissue to that seen in mice carrying human OC transgenes, using immunocytochemistry and in situ hybridization. These results are intended to identify precisely when and where OC is expressed. The second Aim will test the importance of suspected regions within the OC promoter by creating new transgenic lines which carry hOC-CAT transgenes that have these regions mutated, deleted or multimerized. Corresponding studies will be performed in osteoblastic cell lines stably transfected with different OC promoter constructs. Once the OC promoter region(s) comprising within hundred base pairs or less, which confer bone specificity, are identified, attempts will be made to identify putative transacting protein(s). DNase-1 footprinting will also be used to map cis-acting bone-specific sites in transfected cells. It is suggested by the applicants that these assays will position them to eventually isolate the putative proteins(s) using DNA affinity chromatography. Definition of the BSE(s) within the osteocalcin gene will greatly improve our understanding of molecular mechanisms controlling the tissue-specific and developmentally-dependent gene expression. In addition, it will provide a molecular basis by which to target bone active receptors or growth factors to osteoblasts.